Because the invention, which largely solves the problem of enhancing the bioavailability of some bioactive compositions, is based on mint extract byproducts, we start our discussion by providing some background on mint extraction before explaining the nature of the problems solved by the invention.
Mint flavoring has been used in some medications and mint has often been associated with soothing foods, but mint has not been proposed as an adjuvant for bioactive compositions as we have found for a particular fraction of the extracted oils which is freed of most of the mint flavoring compounds.
The mint family of herbs, botanical family name Labiatae, encompasses multiple genera including Rosmarinus (rosemary), Monarda (horsemint) and Mentha (mint). Common mint species include Mentha piperita (peppermint), Mentha spicata (spearmint), Mentha arvensis (corn mint) and Mentha pulegium (pennyroyal). Mint species are well known for the aromatic volatile essential oils in their foliage. Methods of distillation for extraction and refining of the essential oils for use for their aroma or flavor characteristics in perfumes, flavorings and medicines have long been established. Some of the fractions separated are highly desired, and some are extracted due to their tendency to dilute or alter the more desired flavor compositions.
Mint teas comprised of the entire mint leaf, including cellular components and molecules in addition to the essential oils, have been consumed as food and medicine for many centuries. Mint is claimed to be effective for the reduction of digestive disorders including colic, indigestion, nausea, and stomach cramps and reduction of severity of symptoms from Irritable Bowel Syndrome (McKay, et al. (2006) “A review of the bioactivity and potential health benefits of peppermint tea (Mentha piperita L.)” Phytother. Res. 20 (8):619-633, 2006).
Methods have long been available for the separation of volatile organics from plant material and for the further purification of those oils. Most techniques are in the public domain, and a few are the subjects of patents. For example, Rathbun and Thalheimer described a continuous method for separating volatile organic components from fibrous plant material in U.S. Pat. No. 4,495,033. Further separation of distinct volatile components has been described; e.g., Barcelon, et al., described a method for further purification of mint flavor for the production of high-impact chewing gum (U.S. Pat. No. 5,030,459).
Only a small fraction of low-boiling mint compounds are used in the production of flavors/aromas, and the low boiling fractions are often considered byproducts. Owing to the traditionally recognized solvent capabilities of essential oils, low-boiling fractions of flavor oils are often sold as cleaning agents. For example, a distillation method for producing a cleaning product composed of a high percent of terpenes is described by Komocki, et al., in U.S. Pat. No. 6,153,571 and a low-toxicity cleaning solvent compound composed primarily of monoterpenes is described by Lucas, et al., in U.S. Pat. No. 5,665,690. Essential oils are also a component of a cleaner described in U.S. Patent Publication No. 2005/0192199 A1, to Cartwright, et al.
Mint oils are complex essential oils composed of many diverse individual chemicals that range from hydrophilic to lipophilic. Mint oils have been utilized in food for many centuries and mint oils are Generally Recognized As Safe (GRAS) by the U.S. FDA. Mint flavoring has been used in some medications and mint has often been associated with soothing foods, but mint has not been proposed as an adjuvant for bioactive compositions despite the fact that there are significant needs for adjuvants to facilitate the use of bioactives in the treatment of numerous medical conditions and maladies facing mammals, especially modern man.
Heart disease, diabetes and cancer are major health care problems facing the U.S. and many other Westernized countries. Today, the total annual health care cost for Cardio Vascular Disease (CVD) in the U.S. is expected to be more than $431 billion, and the cost for cancer is expected to exceed $200 billion. Additionally, the total annual cost for diabetes in the U.S. is estimated to be $100-150 billion.
A primary class of drugs used for the reduction of serum cholesterol levels, and subsequent reduction of CVD risk are the statins, however, statins inhibit the formation of farnesyl pyrophosphate, an intermediate in the biosynthesis of Coenzyme Q10 (ubiquinone; CoQ10). CoQ10 is utilized in the mitochondria for energy production. Deficiency of CoQ10 induced by statins may be the cause of ‘statin-induced myopathy’, an often-noted lethargy and malaise associated with statin consumption (Littarru et al. (2007 “Coenzyme Q10 and statins: biochemical and clinical implications” Mitochondrion. 7 Suppl:S168-S174). Consequently many health care practitioners urge CoQ10 supplementation concomitant with statin use (Okello et al. (2009) “Combined statin/coenzyme Q10 as adjunctive treatment of chronic heart failure” Med. Hypotheses 73 (3):306-308). However, the bioavailability (defined as the product of absorption from the intestine as well as the utilization of the specific compound by the target tissue) of CoQ10 is quite low, and oral consumption of CoQ10 may have limited effectiveness.
Resveratrol (trans-3,5,4′-trihydroxystilbene) and lycopene (2,6,10,14,19,23,27,31-octamethyldotriaconta-2,6,8,10,12,14,16,18,20,22,24,26,30-tridecaene) are compounds found in red wine and tomatoes, respectively, that may be protective against chronic disease (Kris-Etherton et al., (2002) “Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer” Am. J. Med. 113 Suppl 9B:71S-88S). Increasing evidence suggests that consumption of resveratrol may be protective against heart disease and lycopene may be protective against prostate cancer. Both compounds may function as an antioxidant, anti-inflammatory agent or by other pathways, but most nutritionists agree that the in vivo significance of these compounds also is limited because poor absorption results in limited bioavailability.
There are many other substances capable of benefiting human health but are of questionable benefit because of limited bioavailability. Their poor absorption is possibly related to difficulty to keep them in solution in the absorptive area of the gut. Overcoming problems with bioavailability could open up many possibilities for reducing the risk of chronic disease by inclusion of these and similar compounds in foods, dietary supplements and/or drugs.
Dermal bioavailability is a relatively simple process that is primarily a function of penetration across the various dermal layers, whereas oral bioavailability is considerably more complex; a function of diffusion across the unstirred water layer, active and passive transport into and out of the absorptive cell, hepatic metabolism and re-excretion into the G.I. tract.
There is evidence that dermal penetration of hydrophobic drugs is enhanced by certain naturally-derived compositions. For example, co-administration of hydrophobic drugs with terpene mixtures has shown some promise. See, for example, Songkro, S., et al. (2009) “Effects of some terpenes on the in vitro permeation of LHRH through newborn pig skin” Pharmazie 64 (2):110-115; N. Dragicevic-Curic, N., et al. (2008) “Topical application of temoporfin-loaded invasomes for photodynamic therapy of subcutaneously implanted tumours in mice: a pilot study” J. Photochem. Photobiol. B 91 (1):41-50; Tas, C., et al., (2007) “In vitro and ex vivo permeation studies of etodolac from hydrophilic gels and effect of terpenes as enhancers” Drug Deliv. 14 (7):453-459; Nokhodchi, A. et al. (2007) “The effect of terpene concentrations on the skin penetration of diclofenac sodium” Int. J. Pharm. 335 (1-2):97-105; Das, M., et al. (2006) “Effect of different terpene-containing essential oils on percutaneous absorption of trazodone hydrochloride through mouse epidermis” Drug Deliv. 13 (6):425-431; Cal, K. (2006) “Skin penetration of terpenes from essential oils and topical vehicles” Planta Med. 72 (4):311-316; Nielsen, J., (2006) “Natural oils affect the human skin integrity and the percutaneous penetration of benzoic acid dose-dependently” Basic Clin. Pharmacol. Toxicol. 98 (6):575-581; Cal, K. et al., (2003) “Cutaneous absorption and elimination of three acyclic terpenes—in vitro studies” J. Control Release 93 (3):369-376; Yamane, M., et al. (1995) “Terpene penetration enhancers in propylene glycol/water co-solvent systems: effectiveness and mechanism of action” J. Pharm. Pharmacol. 47 (12A):978-989; Obata, Y. et al. (1990) “Effect of cyclic monoterpenes on percutaneous absorption in the case of a water-soluble drug (diclofenac sodium)” Drug Des Deliv. 6 (4):319-328; Okamoto, H. et al., (1987) “Enhanced penetration of mitomycin C through hairless mouse and rat skin by enhancers with terpene moieties” J. Pharm. Pharmacol. 39 (7):531-534; Vallette, G. et al. (1952) “Percutaneous absorption of the hydrocarbons of the benzene, cycloalkane, cycloalkene and terpene series” Therapie 7 (2):139-143). Multiple patents exist for dermal delivery systems that include terpenes (U.S. Pat. No. 6,342,208 to Hyldgaard et al.; U.S. Pat. No. 6,132,760 to Hendenstrom et al. Oct. 17, 2000; U.S. Pat. No. 6,723,337 to Song et al.; U.S. Pat. No. 5,240,932 to Morimoto, et al.; and patent publications (2004/0127531 to Lu, et al.; U.S. Patent Publication No. 2004/0033254 to Song, et al.; 2005/0181031 to Saito, et al.) also exist for dermal applications that use extracts or absolutes from plant species that contain terpenes. The increased dermal bioavailability of the above compounds is most likely solely a result of the ability of terpenes to effectively dissolve and keep in solution the compound of interest.
The more complex process of oral bioavailability demands studies that specifically address the process.
Numerous attempts have been made to improve the oral bioavailability of molecules such as CoQ10, especially by formulations that increase the solubility of the CoQ10. CoQ10 has been formulated with various lipids including rice bran oil provided in a soft gel capsule (U.S. Pat. Nos. 6,955,820 and 6,616,942 to Udell, and U.S. Pat. Nos. 7,060,263 B-2, 6,623,734 and 7,273,622 to Udell, et al.), various lipids used to form preliposomes (U.S. Patent Publication No. 2005/0037066 to Chen, et al.) and liposomes (U.S. Pat. No. 5,891,465 to Keller, et al.), various lipids (U.S. Pat. No. 6,855,733 to Udell, et al.), emulsifications with various lipids (U.S. Pat. No. 7,094,804 to Behnam et al.; U.S. Patent Publication No. 2004/0152612 A1 to Supersaxo et al), various lipids and triglycerides (U.S. Patent Publication Nos. 2005/0169988 A1 and 2003/0044474 A1 to Tao, et al.), glyceryl esters (U.S. Pat. No. 6,300,377 to Chopra) and emulsifications including organic acids (U.S. Pat. No. 7,026,361 to Minemura, et al.). Additionally the reduced form of CoQ10 (ubiquinol) has been used in preparations with various lipids (U.S. Pat. No. 6,740,338 to Chopra). Non-lipid approaches to increasing the bioavailability of CoQ10 include bacterial and yeast fermentation in the presence of CoQ10 as described by Chokshi in U.S. Pat. No. 6,806,069 B2, and complexing CoQ10 with cyclodextrin (U.S. Pat. No. 7,030,102) and production of nanoscale CoQ10 in a lipid matrix (U.S. Pat. No. 7,438,903 to Parkhideh, et al.). Most of the solvents utilized are common food grade lipids; however, few food grade lipids function well as solvents of CoQ10 and improvements of bioavailability, if reported, are modest at best.
A method for increasing the bioavailability of CoQ10 by solubilization in d-limonene is disclosed in U.S. Pat. No. 7,273,606 to Fantuzzi, et al. The patent states that CoQ10 can be readily dissolved in the monoterpene d-limonene, a primary component of essential oils isolated from lime, and to a much lesser extent, mint. Limonene is GRAS for use as a flavoring agent, however very small amounts are used as flavors and greater amounts are needed to improve bioavailability of bioactive molecules. Although the data are inconclusive, there are toxicity concerns, including cancer, with d-limonene (DeWitt, C. et al. (2004) “Botanical solvents” Clin. Occup. Environ. Med. 4 (3):445-4-vi; Mally, A., et al. (2002) “Non-genotoxic carcinogens: early effects on gap junctions, cell proliferation and apoptosis in the rat” Toxicology 180 (3):233-248). Thus, the total intake of limonene needed for enhancement of bioavailability of bioactive molecules may make it unsuitable for most oral applications, especially for substances that may be ingested on a daily basis over a prolonged period.
Fantuzzi, et al., (U.S. Pat. No. 7,273,606) assert that limonene, and monoterpenes in general, are responsible for solubilizing CoQ10, and that solubilization in turn increases CoQ10 bioavailability. In the body, bile salts are the physiologic mechanism for emulsification and subsequent absorption of fats. However, the effectiveness of bile salts is a result of their amphiphilic nature; i.e., they possess both hydrophilic and lipophilic characteristics (Neuman (1989) Neuman, M. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions. J. Lipid Res. 30 (5):719-730. Limonene appears to function solely as a solvent of hydrophobic molecules.
Benet, et al., teach that essential oils when co-administered with a pharmaceutical compound can increase the bioavailability of that compound (U.S. Pat. Nos. 5,716,928 and 5,665,386 to Benet, et al.). Further, Benet, et al., uses the term “pharmaceutical compound” to encompass, among others, quinone compounds, and Benet, et al., include oils of peppermint and spearmint in their universe of essential oils. However, Benet, et al., teach that increased bioavailability is a function of (in addition to other factors) metabolism of the compound and that bioavailability may be enhanced when metabolic transformations are inhibited. To that end, they have established as a criterion for enhancing bioavailability, the ability of an oil to inhibit conversion of cyclosporine to hydroxylated products, and in general to inhibit enzymes of the cytochrome P450 3A class. Further Benet et al. have provided data showing inhibition of cyclosporine transformation as evidence for increases in bioavailability.
Mullen, in U.S. Pat. No. 5,824,337 is directed to provision of economical micelles and micelle-like structures which can be used as carriers for protection of substances which are lipids or lipophilic or are soluble or easily dispersed in oils or other lipid-like compounds. Examples of such substances are said to include drugs which are lipophilic such as immunogens or steroids, fragrances, flavorings and nutrients. One example shows that micellular solutions can be made with the oils from mint. However numerous substances capable of producing micelles are not capable of dissolving CoQ10 as well as other lipophilic molecules. Moreover, the substances comprising the micelle itself may interfere with metabolism of the bioactive resulting in even decreased bioavailability. Therefore the ability of crude mint oil to form micelles does not demonstrate dissolution of CoQ10 within the micelle nor does it demonstrate increased absorption and/or decreased metabolism of CoQ10.
Biological systems are complex, and isolation of one component does not teach the function of the system as a whole. Benet, et al., teach that peppermint and spearmint oil are among the essential oils that inhibit cytochrome P450 3A, and from this they assume bioavailability is enhanced. Without a direct measure of absorption/bioavailability, however it is not immediately obvious that the one action results in the second outcome. Indeed, Press, R., et al., (2006; “The effect of P-glycoprotein and cytochrome P450 3a on the oral bioavailability of vinorelbine in mice” Cancer Chemother. Pharmacol. 57 (6):819-825) reported that substantial inhibition of cytochrome P450 3A resulted in only a minimal change in the oral bioavailability of vinorelbine in mice. Therefore, it is essential that claims of increased bioavailability be documented by specific measures of such, rather than inferred from isolated processes. Thus, the teaching of Benet does not teach an actual increase in absorption and/or bioavailability of any compound.
Because we have found that a byproduct mint oil fraction has strong adjuvant properties, we note that there are many reasons why raw mint oil is not suitable as a solvent system for the adjuvant purposes. Raw unfractionated mint oil would require too large a dose for inclusion in many dietary supplement and pharmaceutical applications. In order to be suitable for galenical development of these delivery forms, it must be fractionated and extraneous fractions must be eliminated. Raw mint oil also contains substances which may be unsafe in larger doses (e.g., limonene, pulegone), these too must be reduced or eliminated. Moreover, an adjuvant should not have an overpowering flavor that makes consumption of effective amounts difficult.
In vitro measures of bioavailability are often conducted by using Caco-2 cells, a cell line that is recognized by the food and pharmaceutical industry as an excellent in vitro screen to determine potential oral bioavailability. (Press, B. et al. (2008) “Permeability for intestinal absorption: Caco-2 assay and related issues” Curr. Drug Metab 9 (9):893-900) Caco-2 cells are cells derived from a human colon cancer malignancy. If properly maintained, the cells will remain alive indefinitely (i.e. they are immortal), but when grown under correct conditions they can be “transformed” into a cell type that grows to maturity then dies (thus mimicking the way most cells in the body grow). Interestingly, during this transformation the cells transform into a cell almost identical to the absorptive area of the small intestine, and this quality has made the Caco-2 cell line the method of choice for studies of processes that occur in the gut; e.g., absorption.
Absorption of a substance involves two distinct mechanisms—first, the substance must be taken up from the lumen of the gut and enter the absorptive cell, and second, the substance must be excreted from the absorptive cell into the lymph or blood on the serosal side of the cell. A test substance can be added to the medium in which the cells are grown, then the cells can be separated from the medium and the amount of substance that is taken up into the cell can be measured. Additionally, if Caco-2 cells are grown on a porous membrane at the bottom of a small plastic cup, the cells will interlock in ‘tight junctions’ with neighboring cells, resulting in a confluent, water-tight monolayer of cells. This insert can then be placed in a solid well and media put inside the insert (on the apical side of the cells, or the side corresponding to the gut lumen) and in the well (on the basolateral side of the cell or the side corresponding to the serosa of the body). Substances added to the top (apical) side of these cells can only pass to the bottom by being taken up into the cell and then transported (or diffused) out the bottom (basolateral side). Such a system gives a very reliable indication of whether, and to what extent, absorption is taking place; hence an indication of ‘bioavailability’.
Rodent animal studies give a more reliable measure of bioavailability. Increased concentration of the metabolite of interest in the plasma or serum is an accepted measure of bioavailability. However, increased serum concentrations are not necessarily definitive proof that the concentration of the metabolite increases in the tissue (and/or sub-cellular compartment) of interest. CoQ10 is needed for energy production and thus is needed in the mitochondria of tissues that utilize a great amount of energy; the mitochondria of the heart and liver are such tissues (Ochoa, J. et al. (2007) “Effect of lifelong coenzyme Q10 supplementation on age-related oxidative stress and mitochondrial function in liver and skeletal muscle of rats fed on a polyunsaturated fatty acid (PUFA)-rich diet” J. Gerontol. A Biol. Sci. Med. Sci. 62 (11):1211-1218; Kelso, G. et al. (2002) “Prevention of mitochondrial oxidative damage using targeted antioxidants” Ann. N.Y. Acad. Sci. 959:263-274).
There is a present need for methods and compositions effective for enhancing the bioavailability of poorly bioavailable compositions that impart health benefits such as Coenzyme Q10 (ubiquinone; CoQ10), carotenoids (e.g., β-carotene), curcuminoids, lycopene and resveratrol.